C/D box small nucleolar RNA SNORD104 promotes endometrial cancer by regulating the 2ʹ-O-methylation of PARP1

Background Small nucleolar RNAs (snoRNAs) are dysregulated in many cancers, although their exact role in tumor genesis and progression remains unclear. Methods The expression profiles of snoRNAs in endometrial cancer (EC) tissues were analyzed using data from The Cancer Genome Atlas, and SNORD104 was identified as an upregulated snoRNA in EC. The tumorigenic role of SNORD104 in EC was established in CCK8, colony formation, EdU, apoptosis, Transwell, and in vivo xenograft experiments. The molecular mechanisms of SNORD104 were analyzed by RNA immunoprecipitation (RIP), Nm-seq, RTL-P assay, RNA stability assay, qRT-PCR, and western blotting. Results Antisense oligonucleotide (ASO)-mediated knockdown of SNORD104 in Ishikawa cells significantly inhibited their proliferation, colony formation ability, migration, and invasion in vitro and increased apoptosis. On the other hand, overexpression of SNORD104 promoted EC growth in vivo and in vitro. RIP assay showed that SNORD104 binds to the 2ʹ-O-methyltransferase fibrillarin (FBL), and according to the results of Nm-seq and RTL-P assay, SNORD104 upregulated PARP1 (encoding poly (ADP-ribose) polymerase 1) 2ʹ-O-methylation. The binding of FBL to PARP1 mRNA was also verified by RIP assay. Furthermore, SNORD104 expression was positively correlated with PARP1 expression in EC tissues. In the presence of actinomycin D, SNORD104 increased the stability of PARP1 mRNA and promoted its nuclear localization. Finally, silencing FBL or PARP1 in the HEC1B cells overexpressing SNORD104 inhibited their proliferative and clonal capacities and increased apoptosis rates. Conclusions SNORD104 enhances PARP1 mRNA stability and translation in the EC cells by upregulating 2ʹ-O-methylation and promotes tumor growth. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-022-03802-z.


Introduction
Endometrial cancer (EC) is one of the most common malignancies in menopausal and postmenopausal women in developed countries, and its incidence is increasing worldwide [1,2]. Although surgery, chemotherapy, and radiotherapy have achieved satisfactory outcomes, the survival rate of EC patients is still dismal due to the tendency of the tumor cells to relapse and metastasize [3]. Therefore, it is crucial to elucidate the molecular mechanisms underlying the initiation, progression, and metastasis of EC in order to identify potential therapeutic targets for personalized treatment. Small nucleolar RNAs (snoRNAs) are a class of small non-coding RNAs (ncRNAs) with conserved structural elements that are widely distributed in the nucleolus of eukaryotic cells and Page 2 of 14 Lu et al. Journal of Translational Medicine (2022) 20:618 broadly classified into the C/D box snoRNAs (SNORDs) and H/ACA box snoRNAs [4]. They are usually encoded by intron regions of coding genes or non-coding genes and are complicated by the transcription and processing of host genes, with only a few being encoded by independent genomic locations [5]. The snoRNAs were long considered to be involved in the nucleolar function. Recent studies show that snoRNAs mediate 2ʹ-Omethylation and pseudouridylation of rRNA, tRNA, mRNA, snRNA, and other RNAs via complementary base pairing and also regulate gene expression by forming snoRNP complexes with ribonucleolar proteins (RNPs) [6,7]. We identified the snoRNAs potentially related to the development of EC by screening the transcriptomic data of The Cancer Genome Atlas (TCGA) database. SNORD104 (located at 17q23.3, also known as U104) was significantly upregulated in the EC tissues, although its role in cancer, particularly EC, remains unclear. Fibrillarin (FBL) is a component of the small nucleolar ribonucleoprotein (snRNP) (including FBL, NOP56, NOP58) that contains N-terminal repeat domains rich in glycine and arginine residues, similar to the FBLs in other species. The central region of FBL resembles an RNA binding domain and contains a common RNP sequence [8]. FBL is a key 2ʹ-O-ribose methylase that catalyzes SNORD-dependent methylation of target RNAs [9]. However, whether FBL-catalyzed 2ʹ-O-methylation plays an important role in EC remains unknown. In this study, we evaluated the oncogenic effects of SNORD104 in EC through established in vitro and in vivo experiments, and explored the molecular mechanisms by 2ʹ-O-methylation sequencing (Nm-seq) and RNA immunoprecipitation.

Human tissue samples
Tumor tissue samples were collected from 71 treatmentnaïve EC patients who underwent surgery at the Third Affiliated Hospital of Guangzhou Medical University. The samples were snap frozen in liquid nitrogen immediately after resection and stored at − 80 °C. The study protocol was approved by the ethics committee of the hospital, and all patients provided written informed consent.

Cell viability assays
The cells were seeded in a 96-well plate at the density of 2000 cells/100 μl per well and transfected with the respective constructs. The CCK8 reagent (Yeasen Biotechnology, Shanghai, China) was added to the cells at 0, 24, 48, and 72 h, and the absorbance of each well was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). The experiment was repeated thrice.

Colony formation assay
The cells were seeded in a 6-well plate at the density of 500 cells/2 ml per well and transfected with the respective constructs once they adhered. After culturing for 7-10 days, the colonies were fixed using formaldehyde for 15 min, washed thrice with PBS, and stained with crystal violet for 10 min. The plates were rinsed with tap water to remove the excess dye and dried, and the number of colonies was counted.

EdU assay
The Ishikawa/HEC1B cells were seeded in 96-well plates, and EdU solution (Thermo Scientific, USA) was added. The cells were incubated for 16 h, and the subsequent steps were followed as per the manufacturer's instructions. The stained cells were observed under a microscope (Olympus, Tokyo, Japan).

Apoptosis assay
Apoptosis was detected by propidium iodide (PI) and fluorescein isothiocyanate (FITC)-Annexin V (BD Pharmingen, San Diego, CA, USA) staining according to the manufacturer's instructions. Briefly, the cells were harvested 48 h after transfection and washed twice with cold PBS. The suspension was stained with 100 μl 1 × buffer and 5 μl FITC-Annexin V and PI in the dark for 15 min. The reaction was terminated by adding 400 μl 1 × buffer, and the cells were analyzed by flow cytometry within 1 h.

Quantitative real-time reverse transcription PCR
Total RNA was extracted from EC cell lines or tissues using TRIzol reagent (1 ml; Takara, Shiga, Japan) and reverse transcribed to cDNA. Quantitative real-time PCR was performed using the SYBR PreMix Ex TAQ II kit (Takara). The primer sequences were as follows: U6:

Western blotting
Total protein was extracted from cultured cells or tissues using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors and quantified using the BCA Protein Assay Kit (Beyotime, China). The protein samples were diluted with the appropriate amount of loading buffer and PBS to 2 μg/l, and denatured by heating at 95 °C for 15 min. The samples were resolved by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham, Munich, Germany). Following overnight incubation with rabbit anti- and washed again with 1% TBST buffer for 10 min. The bands were visualized using an enhanced chemiluminescence system (NCM Biotech, Suzhou, China), and the protein levels were quantified using grayscale values.

RNA immunoprecipitation (RIP) assay
RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) as per the manufacturer's instructions. Cells growing at 80-90% confluence were harvested and lysed using RIPA lysis buffer. The cell extracts were incubated with magnetic beads with anti-mouse FBL (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or normal mouse IgG (negative control) in the RIPA lysis buffer. After digesting the protein using protease K, the immunoprecipitated RNA was isolated and analyzed using qRT-PCR.

Nm-seq analysis
Nm-sequencing was performed by the Shanghai Cloudseq Biotech Co. Ltd. as previously described [12] using the Illumina NovaSeq 6000 sequencer.

Reverse transcription at low dNTPs-PCR (RTL-P) assay
RTL-P assay was performed to detect the 2′-O-methylation level (Nm) of PARP1 according to published methods [13,14]. Briefly, 5 μg total RNA was incubated with low (1 μM) or high (1 mM) concentration of dNTPs (Takara) and 1 μl specific RT primers at 65 °C for 5 min and then placed on ice. The reaction was performed using 4 μl M-MLV RT 5 × buffer (PROMEGA), 1 μl 200 U/μl M-MLV Reverse Transcriptase (PROMEGA), 1 μl 0.1 M DTT (PROMEGA), and 1 μl 40 U/μl RNase inhibitor (PROMEGA) at 50 °C for 1 h, and then at 70 °C for 15 min. QRT-PCR was performed on the SYBR Prime X Ex-TAQ Patent II Suite (Takara) using PARP1-specific primers. The relative expression of the gene was calculated by comparing the period threshold (Ct) of the target gene in the experimental group and the control group at low concentrations according to the 2 −ΔCt method.

RNA stability assay
Stably transfected cells were seeded into a 6-well plate and incubated until they reached 70% confluence. Actinomycin D (Sigma, St. Louis, MO, USA) was added (Act D, 5 µg/ml), and the cells were harvested at 0, 6, and 12 h. Total RNA was extracted using TRIzol reagent, and PARP1 mRNA levels were detected by qRT-PCR.

Nucleocytoplasmic separation assay
The confluent cells were harvested using a scraper and centrifuged at 1500 rpm for 5 min. The supernatant was removed, and the nuclear and cytoplasmic fractions were

Statistical analysis
GraphPad Prism [version 8.02(263)] was used for statistical analysis. Non-parametric Mann-Whitney test for small samples that do not meet the normal distribution; Student's t-test was used for the normal distribution and when the sample size was large; Correlations between groups were determined using the Pearson correlation coefficient. The CCK-8 assay data were analyzed using ANOVA. All data were presented as the mean ± S.D of at least three independent experiments. Statistical significance was set at P < 0.05.

SNORD104 is upregulated in EC tissues
We analyzed the transcriptomic profiles of 548 EC tissue samples and 35 normal endometrial tissue samples from TCGA datasets (Fig. 1A), and found that SNORD104 was upregulated in the EC samples. To verify these results, we analyzed the expression of SNORD104 in post-operative EC tissues collected from the Third Affiliated Hospital of Guangzhou Medical University and analyzed the correlation between the expression of SNORD104 and clinicopathological features (Additional file 1: Table S1). SNORD104 expression was higher in patients with stage III/IV disease (n = 13) compared to those with stage I/II disease (n = 46) (Fig. 1B). In addition, SNORD104 was upregulated in the poorly differentiated (n = 15) versus the moderately or well-differentiated (n = 44) tumors (Fig. 1C). SNORD104 expression was also higher in patients with deep myometrial invasion (n = 26) compared to those with shallow myometrial invasion (n = 33) ( Fig. 1D), as well as in patients with lymph node metastasis (n = 26) relative to those without lymph node metastasis (n = 33) (Fig. 1E). In contrast, there was no significant correlation between SNORD104 expression and vascular invasion (Fig. 1F). Therefore, we hypothesized that SNORD104 functions as an oncogene in EC.

SNORD104 knockdown inhibited the malignant potential of EC cells in vitro
Genome sequence analysis showed that SNORD104 was processed from the first intron region of the primary transcript of the SNHG25 gene (small nucleolar RNA host gene 25, located at 17q21.3) (Fig. 1G), and the mature SNHG25 RNA was transcribed from the exons. We found that SNHG25 was also upregulated in EC tissues (Additional file 2: Fig. S1A, *P < 0.05). In addition, SNORD104 was upregulated in the EC cell lines compared to that in the immortalized endometrial cells. Both SNORD104 and SNHG25 were highly expressed in the Ishikawa cells, but showed relatively low expression levels in the HEC1B and HEC1A cells ( Fig. 2A, Additional file 2: Fig. S1B, *P < 0.05). Previous studies have shown that the host genes of snoRNAs regulate their respective snoRNAs and play a role in tumor development [15,16]. To determine the role of SNORD104 and SNHG25 in EC cells, we respectively transfected the Ishikawa cells with SNHG25-specific small interfering RNA (siRNA) and the HEC1B cells with SNHG25-overexpression plasmid, and an antisense oligonucleotide sequence (ASO) targeting SNORD104 in HEC1B cells. The knockdown efficiency was verified by qRT-PCR (Fig. 2B, Additional file 2: Fig. S1C, D, *P < 0.05). SNORD104 knockdown significantly decreased the viability, proliferation (Fig. 2C, D, *P < 0.05), clonal capacity ( Fig. 2E, P < 0.05), migration, and invasion (Fig. 2G, *P < 0.05) of Ishikawa cells, and increased the apoptosis rates (Fig. 2F, *P < 0.05). In contrast, knocking down or overexpressing SNHG25 had almost no effect on the proliferation and apoptosis of endometrial cancer cells (Additional file 2: Fig. S1E-H, ns means P > 0.05).

SNORD0104 upregulates and stabilizes PARP1 by promoting 2ʹ-O-methylation
The SNORDs and H/ACA box snoRNAs regulate gene expression via 2ʹ-O-methylation and pseudouridylation, respectively. Therefore, we hypothesized that SNORD104 might be involved in FBL-mediated 2ʹ-O-methylation. Indeed, the RIP assay of the lysates from SNORD104overexpressing HEC1B cells using anti-FBL antibodies revealed significant enrichment of SNORD104 in the immuno-precipitate (Fig. 4A, *P < 0.05). However, SNORD104 overexpression did not affect the FBL protein level (Fig. 4B).
To identify potential target genes regulated by SNORD104-mediated 2ʹ-O-methylation, we performed NM-seq in the control and SNORD104-overexpressing cells. As shown in Fig. 4C, 2ʹ-O-methylation was upregulated (fold change ≥ 2) at 928 methylated sites (671 RNAs). It's reported small nucleolar RNAs generally guide the modification of target RNAs through complementary base pairing. Therefore, we intersect the RNAs with up-regulated 2ʹ-O-methylation levels and the RNAs with complementary base pairing fragments of SNORD104. Next, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these hypermethylated RNAs. And the results showed that the apoptosis-related pathway ranked first (Fig. 4C). Further, we analyzed the enriched RNAs in apoptosis pathways, and the CPTAC database showed that their encoded proteins were upregulated in endometrial cancer tissues, including ITPR3, LMNB1, PARP1, PARP4, and TUBA1C (Additional file 3: Fig.  S2A). Next, we used RIP assay to detect whether FBL protein binds to these 5 RNAs, and western blot to detect whether the proteins encoded by these 5 RNAs changed.
The results show that only PARP1 mRNA binds to FBL protein (Fig. 4D, *P < 0.05), and only PARP1 mRNA and protein level changes (Fig. 4E, F, *P < 0.05). Therefore, we next analyzed the expression of PARP1 in post-operative EC tissues and observed a positive correlation between SNORD104 and PARP1 expression levels (Fig. 4G, *P < 0.05). PARP1 is primarily localized in the nucleus and is involved in DNA damage repair and ribosome biogenesis. Consistent with this, SNORD104 overexpression increased PARP1 protein levels in the nuclear fraction (Fig. 4H). In addition, the SNORD104-overexpressing xenografts also showed increased PARP1 protein levels ( Fig. 4I, J). RTL-P assay was next performed to detect the level of 2ʹ-O-methylation in PARP1 mRNA (Fig. 5A). SNORD104 knockdown and overexpression respectively decreased and increased the 2ʹ-O-methylation level of PARP1 mRNA (Fig. 5B, C, *P < 0.05). Furthermore, FBL knockdown in the SNORD104-overexpressing HEC1B cells decreased both 2ʹ-O methylated PARP1 mRNA as well as PARP1 protein levels (Fig. 5D, F, *P < 0.05). The biological role of ribose 2ʹ-O-methylation in mRNA is still unclear, although there are reports that it can improve the stability of nucleic acids against alkaline or enzymatic hydrolysis, most likely by altering their physical and chemical properties [17,18]. To test this hypothesis, we treated the SNORD104-overexpressing or knockdown cells with transcription inhibitor actinomycin D. Compared to the control group, the SNORD104-overexpressing cells had significantly higher PARP1 mRNA stability (Fig. 5G, *P < 0.05), while knocking down either SNORD104 or FBL reduced the stability of PARP1 mRNA (Fig. 5H, I, *P < 0.05) (Additional file 3).

Discussion
Endometrial cancer (EC) occurrence and progression involve multiple genetic mutations and epigenetic changes, along with long-term stimulation of estrogen [21,22]. In recent years, various non-coding RNAs, including long non-coding RNAs, circular RNAs, and small non-coding RNAs, such as microRNAs, piwi-interacting RNAs, and snoRNAs, have been identified that regulate tumor epigenetics. snoRNAs are dysregulated in multiple cancer and are associated with tumor cell growth, metastasis, and self-renewal [23]. For example, SNORD42A promotes acute myeloid leukemia cell proliferation by directing 18S rRNA 2'-O-methylation that promotes protein translation [24]. SNORD89 is highly expressed in ovarian cancer stem cells and promotes dryness of ovarian cancer cells by regulating the Notch1-c-Myc pathway [25]. SNORA74A activates PARP1-directed DExD-box helicase 21 (DDX21) ADP-ribosylation and increases the nuclear localization of DDX21 protein, thereby promoting ribosomal biogenesis and cancer cell growth [26]. However, little is known regarding the role of snoRNAs in EC. To identify the regulatory factors that play a role in the development of EC, we screened for the differentially expressed genes in EC tissues relative to the normal endometrial tissues using the Human Genome Map TCGA Database, and found that SNORD104 and its parent gene SNHG25 were significantly upregulated in EC tissues. Although the expression levels of a few snoRNAs correlate with that of their host genes [27], the expression and biological functions of most snoRNAs seem to be independent of their host genes [28,29]. In this study, we found that SNORD104, rather than its host gene SNHG25, promotes EC progression. SNORD104 increased the malignant potential of the EC cells in vitro and in vivo, and inhibited apoptosis, thus preliminarily confirming its pro-oncogenic effects in EC (Fig. 7). The 2ʹ-O-methylation is a relatively conserved type of post-transcriptional modification that is abundant in rRNAs and tRNAs, and protects them against degradation [30]. High-throughput deep sequencing has revealed that 2ʹ-O-ribose methylation exists in the mRNAs and sncRNAs as well [31,32]. In the present study, RIP experiments showed that SNORD104 binds to FBL protein without affecting the latter's expression levels, which further suggests that the biological function of SNORD104 may be exerted via the 2ʹ-O-ribose methylation catalyzed by FBL.
To further confirm this hypothesis, we performed NM-seq in HEC1B cells that overexpressed SNORD104 and identified 928 upregulated 2ʹ-O-methylation sites (671 RNAs). Since snoRNA mainly regulates the modification of target RNAs through complementary base pairing, we intersect the two data sets of Nm-seq and SNORD104-Blast. KEGG pathway enrichment analysis of these RNAs identified that the apoptosis pathway was significantly enriched. Then we analyzed the RNAs enriched in the apoptotic pathway through CPTAC database, and showed that the protein level of ITPR3, LMNB1, PARP1, PARP4, and TUBA1C were upregulated in endometrial cancer tissues. Next, RIP assays confirmed that FBL could bind to PARP1 mRNA, but not the other 4 RNAs, and SNORD104 expression was positively   [35], increasing RNA stability [36], and regulating RNA transport [37]. To determine the mechanism by which SNORD104-mediated 2ʹ-O-methylation upregulates PARP1 protein, we assessed PARP1 mRNA stability after treatment with actinomycin D. SNORD104 overexpression or repression significantly enhanced or reduced PARP1 mRNA stability, respectively. In addition, in HEC1B cells overexpressing SNORD104, FBL knockdown reduced the PARP1 protein levels. This suggested that SNORD104 formed a complex with FBL to target and regulate 2ʹ-O-methylation of PARP1, thereby increasing its protein level and enhancing its biological function. Finally, to verify that SNORD104 exerts its carcinogenic role by regulating the expression of PARP1, we knocked down FBL and PARP1, respectively, in SNORD104-overexpressing HEC1B cells, which expectedly reversed the oncogenic effects of SNORD104 in EC.